There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical, and biological substances. Of particular value are methods for measuring small quantities of pharmaceuticals, metabolites, microorganisms, and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, antibodies, metabolites, enzymes and nucleic acids.
The presence of these materials can often be determined by binding methods which exploit the high degree of specificity which characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable "label" which has been attached to one or more of the complexing materials.
The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting a material of interest. A preferred label should be inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without changing the important binding characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging from hours to months. Detection of the label should be rapid, sensitive, and reproducible without the need for expensive, specialized facilities or personnel. Quantification of the label should be relatively independent of variables such as temperature and the composition of the mixture to be assayed. Most advantageous are labels which can be used in homogeneous systems, i.e., systems in which separation of the complexed and uncomplexed labeled material is not necessary. This is possible if the detectability of the label is modulated when the labeled material is incorporated into a specific complex.
A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are quite versatile, and can be detected at very low concentrations. However, they are expensive, hazardous, and their use requires sophisticated equipment and trained personnel. Furthermore, the sensitivity of radioactive labels is limited by the fact that the detectable event can, in its essential nature, occur only once per radioactive atom in the labeled material. Moreover. radioactive labels cannot be used in homogeneous methods.
Thus, there is wide interest in non-radioactive labels. These include molecules observable by spectrophotometric, spin resonance, and luminescence techniques, as well as enzymes which produce such molecules. Among the useful non-radioactive labeling materials are organometallic compounds. Because of the rarity of some metals in biological systems, methods which specifically assay the metal component of the organometallic compounds can be successfully exploited. For example, Cais, U.S. Pat. No. 4,205,952 (1980) discloses the use of immunochemically active materials labeled with certain organometallic compounds for use in quantitating specific antigens. Any general method of detecting the chosen metals can be used with these labels, including emission, absorption and fluorescence spectroscopy, atomic absorption, and neutron activation. These methods often suffer from lack of sensitivity, can seldom be adapted to a homogeneous system, and as with atomic absorption; sometimes entail destruction of the sample.
Of particular interest are labels which can be made to luminesce through photochemical, chemical, and electrochemical means. "Photoluminescence" is the process whereby a material is induced to luminesce when it absorbs electromagnetic radiation. Fluorescence and phosphorescence are types of photoluminescence. "Chemiluminescent" processes entail the creation of the luminescent species by a chemical transfer of energy. "Electrochemiluminescence" entails the creation of the luminescent species electrochemically.
These luminescent items are of increasing importance. For example, Mandle, U.S. Pat. No. 4,372,745 (1983) discloses the use of chemiluminescent labels in immunochemical applications. In the disclosed systems, the labels are excited into a luminescent state by chemical means such as by reaction of the label with H.sub.2 O.sub.2 and an oxalate. In these systems, H.sub.2 O.sub.2 oxidatively converts the oxalate into a high energy derivative, which then excites the label. This system will, in principle, work with any luminescent material that is stable in the oxidizing conditions of the assay and can be excited by the high energy oxalate derivative. Unfortunately, this very versatility is the source of a major limitation of the technique; typical biological fluids containing the analyte of interest also contain a large number of potentially luminescent substances that can cause high background levels of luminescence.
Another example of the immunochemical use of chemiluminescence which suffers from the same disadvantages is Oberhardt et al., U.S. Pat. No. 4,290,815, (1981) who disclose the in situ electrochemical generation of an oxidant (e.g., H.sub.2 O.sub.2) in close proximity to an immunoreactant labeled with a chemiluminescent species. The electrogenerated oxidant diffuses to the chemiluminescent species and chemically oxidizes it, resulting in the net transfer of one or more electrons to the electrogenerated oxidant. Upon oxidation, the chemiluminescent species emits a photon. In contrast, the subject invention requires the direct transfer of electrons from a source of electrochemical energy to a chemiluminescent species which is capable of repeatedly emitting photons.
The present invention is concerned with electrochemiluminescent labels. Suitable labels comprise electrochemiluminescent compounds, including organic compounds and organometallic compounds. Electrochemiluminescent methods of determining the presence of labeled materials are preferred over other methods for many reasons. They are highly diagnostic of the presence of a particular label, sensitive, nonhazardous, inexpensive, and can be used in a wide variety of applications.
Organic compounds which are suitable electrochemical labels include, for example, rubrene and 9,10-diphenyl anthracene. Many organometallic compounds are suitable electrochemical labels. For instance, Bard et al., in U.S. Pat. No. 5,310,687 (1994), discloses that a wide variety of analytes of interest and chemical moieties that bind to analytes of interest may be conveniently attached to ruthenium- or osmium-containing labels through amide or amine linkages. The labeled materials may then be determined by any of a wide variety of means, including photoluminescent, chemiluminescent, and electrochemiluminescent means. It is also disclosed therein that electrochemiluminescent labels, including ruthenium- and osmium-containing labels and organic molecules such as rubrene and 9,10-diphenyl anthracene, are particularly versatile and advantageous.
The series of elements having Atomic Numbers in the range 57-71 are known as the lanthanides; they are: cerium, dysprosium, erbium, europium, gadolinium, holmium, lanthanum, lutetium, neodymium, praseodymium, promethium, terbium, thulium, and ytterbium. The lanthanides are classically referred to as the `rare earths`. They are also referred to as the inner-transition elements, because outer electron structure is identical across the group; their electron structures differ only at an inner level. Since the electronic diversity between the atoms is at some depth, the elements are very similar chemically. All of the lanthanides form trivalent ions and complexes. Their absorption bands are narrow compared with those of the normal transition ions. Complexing agents, which alter the absorption spectra of normal transition ions by modifying their outer electron structures, have little effect on the lanthanide ions. In spite of a high charge, lanthanide ions are too large to cause significant polarization, so complex formation is not facile.
With the transition metal chelates, the center metal atoms are unfulfilled d-orbitals. Under the ligand field, d-orbitals interact with the ligand field leading to splitting the d-orbitals into two different energy levels, which correspond to the ground state and to an excited state. Unlike the case with transition metals, rare earth metals have unfulfilled f-orbitals. The emission of rare earth metal chelates is due to the intromolecular energy transfer from the ligand to the metal ion. Therefore, the emission is more characteristic of the metal than of the interaction between the metal and its ligands.
The rare earth metal chelated usually have eight coordination sites rather than six as for transition metal chelates. The eight coordination sites are arranged as four in one plane and the other four in another plane, with the metal located between the planes. However, the two planes twist 45.degree. with respect to each other, so this type of compound is not of an octahedral configuration.
The Bard et al. chemical moieties as disclosed in U.S. Pat. No. 5,310,687 do have one characteristic that constitutes a drawback in certain circumstances. The emission spectra of their chelates has a band width on the order of 100 nm. This can make signal discrimination difficult in multiple wavelength electrochemiluminescence measurements. Thus there remains a need for improvement in this area.